Microencapsulation is the process of enveloping certain drugs, enzymes, toxins, or other substances in polymeric matrices. It can be used in controlled release or delayed release of drugs. The many applications, available matrices, and techniques are extensively covered elsewhere (see, for example, Chang, T. M. S. [1977] Biomedical applications of immobilized enzymes and proteins, Vols. 1-2, New York: Plenum Press; Deasy, P. B. (ed.) [1984] "Microencapsulation and related drug processes," In J. Swarbrick (ed.), Drugs and the pharmaceutical sciences: Vol. 20. Microencapsulation and related drug processes, New York: Marcel Dekker, Inc.; McGinity, J. W. [1989] "Aqueous polymeric coatings for pharmaceutical dosage forms," Drugs and the Pharmaceutical Sciences 36; Nixon, J. R. (ed.) [1976] "Microencapsulation," In J. Swarbrick (ed.) Drugs and the pharmaceutical sciences: Vol. 3, New York, Marcel Dekker, Inc.).
Polymeric matrix microencapsulation of microorganisms is a relatively new technology which has potentially major implications in the treatment of various afflictions. Examples of afflictions in which treatment involving microcapsules could be advantageous are diabetes and urinary stone diseases. Insulin dependent diabetes mellitus (IDDM) is a severe disease which afflicts millions of Americans, causing substantial disruption of lifestyle and often resulting in severe health problems. The exact causes of IDDM have remained largely a mystery, despite years of intensive research on this disease. It is now widely recognized that IDDM is an autoimmune condition whereby the body's natural immunological defenses destroy the .beta.-cells of the pancreas. .beta.-cells are responsible for the production of insulin, and, once a substantial portion of the .beta.-cells are destroyed, those individuals afflicted with the disease must rely on exogenous sources of insulin, usually in the form of injections. The success of pancreas or islet cell transplantations is very limited because of immune responses typically mounted by the recipient against the foreign cells.
Urolithiasis, or urinary stone disease, is a common urinary tract problem afflicting more than 10% of the U.S. population. Urinary tract stones are usually classified according to their composition, with the most frequently encountered (70%) being the calcium stone composed of calcium oxalate alone or calcium oxalate mixed with calcium phosphate. Although precipitation of calcium oxalate depends on a urine saturated with both calcium and oxalate ions in a metastable state, it has been argued that the oxalate ion concentration is more significant in the formation of urinary calcium oxalate stones. Thus, the management of oxalate in individuals susceptible to urolithiasis would seem especially important. The majority of oxalate in plasma and urine is derived from the endogenous metabolism of ascorbic acid, glyoxylate, and to a lesser degree, tryptophan. In addition, between 10% and 20% of the urinary oxalate is absorbed from the diet, especially through ingestion of leafy vegetables and plant materials, although there is disagreement in the literature about the relative amounts of diet and endogenous oxalate. Ingestion of ethylene glycol, diethylene glycol, xylitol, and excess ascorbic acid can lead through metabolic conversions to disorders of excess oxalate. Use of methoxyfluraneas an anaesthetic can also lead to oxalosis. Aspergillosis, infection with an oxalate-producing fungus, can lead to production and deposition of calcium oxalate. Other causes of excess oxalic acid include renal failure and intestinal disease.
It is believed that lowering the oxalate levels in the plasma, and subsequently the urine, would decrease the incidence of calcium oxalate stone formation. Unfortunately, there are no known naturally occurring oxalate degrading or metabolizing enzymes in vertebrates. Catabolism of oxalic acid appears restricted to the plant kingdom.
Hyperoxaluria can also be related to genetic disorders. Primary hyperoxaluria is a general term for an inherited disorder which reveals itself in childhood and progresses to renal failure and frequently death in adolescence. It is characterized by high urinary excretion of oxalate and recurring calcium oxalate kidney stones. Primary hyperoxalurias consist of two rare disorders of glyoxylate and hydroxypuruvate metabolism. There are no satisfactory treatments for the two types of primary hyperoxaluria. Hemodialysis and renal transplantation have not been successful in halting the progress of this disease. Controlled diet has also failed to stop the complications of primary hyperoxaluria. Primary hyperoxaluria eventually leads to other abnormalities such as urolithiasis, nephrocalcinosis with renal failure, systemic oxalosis, and oxalemia.
Oxalate toxicity can also cause livestock poisoning, due to grazing on oxalate-rich pastures. Ingestion of oxalate-richplants such as Halogeton glomeratus, Bassia hyssopifolia, Oxalis pes-caprae, and Setaria sphacelata, or grains infected with the oxalate-producing fungi Aspergillus niger, has been reported to cause oxalate poisoning in sheep and cattle. Chronic poisoning is often accompanied by appetite loss and renal impairment. Acute toxicity can lead to tetany, coma, and death (Hodgkinson, A. [1977] Oxalic acid in biology and medicine, London: Academic Press, pp. 220-222).
Three mechanisms for oxalate catabolism are known: oxidation, decarboxylation, and activation followed by decarboxylation (Hodgkinson, A. [1977], supra at 119-124). Oxalate oxidases are enzymes that are found in mosses, higher plants, and possibly fingi which catalyze the oxidation of oxalate to hydrogen peroxide plus carbon dioxide: (COOH).sub.2 +O.sub.2 .fwdarw.2CO.sub.2 +H.sub.2 O.sub.2. Oxalate decarboxylases are enzymes which produce CO.sub.2 and formate as products of oxalate degradation. An O.sub.2 -dependent oxaiate decarboxylase found in fungi catalyzes the decarboxylation of oxalic acid to yield stoichiometric quantities of formic acid and CO.sub.2 : (COOH).sub.2 .fwdarw.CO.sub.2 +HCOOH. Varieties of both aerobic and anaerobicbacteria can also degrade oxalic acid. An activation and decarboxylation mechanism is used for degradation of oxalate in Pseudomonas oxalaticus and other bacteria. The many pathways leading to oxalate are discussed elsewhere (Hodgkinson, A. [1977] supra; Jacobsen, D. et al. [1988] American Journal of Medicine 84:145-152).
Oxalobacter formigenes is a recently described oxalate-degrading anaerobic bacterium which inhabits the rumen of animals as well as the colon of man (Allison, M. J. [1985] Arch. Microbiol. 141:1-7). O. formigenes OxB is a strain that grows in media containing oxalate as the sole metabolic substrate. Other substrates do not appear to support its growth. The degradation of oxalate catalyzed by the bacterial enzyme results in CO.sub.2 and formic acid production (Allison [1985], supra).
Recently, research has focused on matrices used to encapsulate cells and organisms. The use of alginate gel technology to formulate agricultural products, pesticides, and food items has been disclosed. For example, U.S. Pat. No. 4,053,627 describes the use of alginate gel discs for mosquito control; U.S. Pat. No. 3,649,239 discloses fertilizer compositions; and U.S. Pat. No. 2,441,729 teaches the use of alginate gels as insecticidal as well as candy jellies. In addition, U.S. Pat. Nos. 4,401,456 and 4,400,391 disclose processes for preparing alginate gel beads containing bioactive materials, and U.S. Pat. No. 4,767,441 teaches the use of living fungi as an active material incorporated in an alginate matrix.
The most usual hydroxyl polymers used for encapsulating biomaterials are alginate, polyacrylamide, carrageenan, agar, or agarose. Of these, alginate and carrageenan are the only ones which can be manufactured simply in spherical form with encapsulated material. This is done by ionotropic gelling, i.e., the alginate is dropped down into a calcium solution and the carrageenan into a potassium solution. However, the resulting beads are stable only in the presence of ions (calcium and potassium, respectively).
The use of ultrasonic nozzles has offered a new way of making smaller microspheres with very good control over the size of the droplets (Ghebre-Sellassie, I. [1989] "Pharmaceutical pelletilization technology," In J. Swarbrick (ed.) Drugs and the pharmaceutical sciences: Vol. 37. Pharmaceutical pelletilization technology, New York: Marcel Dekker). Liquid is supplied at low pressure and droplets are formed at the tip of the nozzle by ultrasonic frequency. However, it has not been possible to atomize the higher viscosity alginates.
Cellulose acetate phthalate (CAP) is a polyelectrolyte containing ionizable carboxyl groups. It is an enteric coating widely used in the industry for coating tablets. Enteric coatings protect the drug from the gastric juices (pH range 1-6) (Yacobi, A., E. H. Walega [1988] Oral sustained release formulations: Dosing and evaluation, Pergammon Press). CAP serves this purpose by being virtually insoluble below pH 6.0. Aquateric is a commercially available pseudolatex with CAP content of .apprxeq.70%. Other constituents include Pluronic F-68, Myvacet 9-40, polysorbate 60 and .ltoreq.4% free phthalic acid (McGinity [1989], supra). Both CAP and aquateric can be fabricated into microspheresby first dissolving them in pH 7.0 distilled deionized water and dropped in acidic solution (Madan, P. L., S. R. Shanbhag [1978] Communications, J. Pharmac. 30:65). Others have used coacervation as the method for microencapsulation (Merkle, H. P., P. Speiser [1973] J. Pharmac. Sci. 62:1444-1448).
The use of various matrices to encapsulate cells and organisms for implantation in the body has been previously reported (Sun, A. M. [1988] "Microencapsulation of pancreatic islet cells: A bioartificial endocrine pancreas," In Mosbach, K. (ed) Methods in enzymology: Vol 137, Academic Press, Inc.). Pancreatic cells have been utilized in vitro and in vivo for the production and delivery of insulin. Long term in vivo (in rats) studies of alginate microcapsules containing islet cells, implanted in the peritoneal cavity, have shown great biocompatibility with no cell adhesion to the capsules and a reversal to normal of the previously diagnosed diabetic rats (Sun, A. M., Z. Cai, Z. Shi, F. Ma, G.M. O'Shea [1987] Biomaterials, Artificial Cells, and Artificial Organs 15(2):483-496).
In vitro cell cultures of hybridomas are now routinely utilized for the preparation of monoclonal antibodies of great specificity. Cancer cell lines are used in vitro for formation of such hybridomas, and also for the screening and testing of potential carcinogenic and anticarcinogenic compounds. Also, the industrial utilization of isolated immobilized cells has received attention, since these can be used as catalysts for biochemical reactions, and such reactions can be used as important tools in syntheses and analytical determinations.
In many instances, the direct introduction of a foreign cell into a host can produce severe immune response in the host. For example, when growing hybridoma cells in the ascites fluid of a host such as a mouse, the mouse has to be pretreated to prevent immune response. When injecting whole islet cells into a human, immune response is also a complicating factor. A need therefore exists for improved methods of facilitating the introduction of such cells into a host, as well as generally for facilitating the manipulation of cells in vitro. The problems confronted by the practitioner in attempting to extend many of the prior art techniques to the encapsulation of living cells or other sensitive biomaterial are numerous. Many of the existing techniques operate under conditions which are too drastic for the survival or continuing viability of a living cell, or cause degradation of the biomaterial desired to be encapsulated. For example, the use of organic solvents, high temperatures, reactive monomers, cross linking conditions, and the like, may hamper the viability or otherwise degrade the biomaterial to be encapsulated. Moreover, it is crucial to prevent dehydration or osmotic rupture of the cell. Another serious problem is the necessity of providing the microcapsule walls with sufficient permeability for nutrients, and secretion and excretion products, to pass through, yet prevent the entry of molecules or cells of a host, for example, products of the hosts immune response, which could destroy the encapsulated material. A further complicating factor is the need to provide sufficient structural integrity of the capsule while keeping the above considerations in mind. Prior art methods of alginate encapsulation, while gentle to the encapsulated material, have failed to produce a capsule of sufficient strength to maintain structural integrity over a long period of time.
A need therefore continues to exist for a method to encapsulate living cells and other sensitive biomaterial under sufficiently mild conditions which allow the cells or biomaterial to remain substantially unaffected by the encapsulation process, yet which also allow the formation of a capsule of sufficient strength to exist over long periods of time.